Archaeal histones share a common ancestry with the histone-folds of the eukaryotic nucleosome cores histones and subunits of many large eukaryotic transcription factors. The histone fold is a structural motif that directs dimer formation, and archaeal histones form homodimers and heterodimers but eukaryotic histone folds now form only heterodimers. Archaeal histones also bind DNA and assemble into complexes spontaneously and individually, and can wrap DNA in either a negative or positive supercoil. They therefore provide a unique opportunity, and a practical experimental system to establish the molecular determinants of histone:histone and histone:DNA interactions. The experiments proposed will generate a detailed structural understanding of histone fold partner specificity, histone-DNA affinity, the positioning of histone assembly, and the determinants of the direction of DNA supercoiling. These are all issues of central and fundamental importance to understanding genome organization and the roles of histones and histone foldcontaining complexes in regulating gene expression in Eukaryotes, and in Archaea. The information gained will also be of importance in understanding how defects in these molecular assemblies and interactions negatively impact human development and health. We have established that different archaeal histone assemblies have sequence-dependent differences in DNA affinity, and this suggests an entirely novel concept for gene regulation based on alternative histone dimer assembly. Chromatin immunoprecipitation coupled with DNA microarray hybridization will be used to investigate this new idea, and to test the hypothesis that this archaeal histone regulatory system was the foundation for eukaryotic genome silencing and gene regulation by positioned nucleosome assembly.